Acute and chronic liver infections caused by Hepatitis B virus (HBV) constitute a major worldwide public health crisis affecting nearly 2 billion people including 1.7 million in the US (WHO report). There are an estimated 350 million chronic carriers of HBV worldwide. According to the Centers for Disease Control, nearly 3 to 7 million people die each year from complications associated with the infection such as cirrhosis of the liver and hepatocellular carcinoma. Significant numbers of liver transplant recipients have continued needs for effective anti-HBV therapy. HBV is recognized as an important etiological agent that causes significant number of human cancers. HBV infection also leads to fulminant hepatitis, a fatal disease in which the liver is destroyed. Chronic hepatitis infection leads to chronic persistent hepatitis, fatigue, liver cirrhosis, liver cancer and death. The epidemiology of HBV infection is similar to that of human immunodeficiency virus (HIV). Many HIV carriers are co-infected with HBV. However, HBV is 100 times more infectious than HIV.
Although three anti-HBV drugs have been currently approved for clinical use, significant unmet medical need exists due to rapid emergence of resistance, and dose-limiting toxicity associated with therapy. The drugs approved for clinical use includes alpha interferon, a genetically engineered protein, and nucleoside analogs such as lamivudine, and entacavir. Another approved anti-HBV drug is adefovir dipivoxil, which is considered a mononucleotide phosphonate analog.
A number of synthetic nucleosides are being developed as anti-HBV agents. For example, the (−)-enantiomer of BCH-189 (2′3′-dideoxy-3′-thiacytidine), known as Lamivudine or 3-TC, is claimed by Liotta et al in U.S. Pat. No. 5,530,116.
FTC or Beta-2-hydroxymethyl-5-(5-fluorocytosin-1-yl)-1,3-oxathiolane claimed by Liotta et al., U.S. Pat. Nos. 5,814,639 and 5,914,331. See also Furman et al., Antimicrobial Agents and Chemotherapy, 2686-2692, 1992. L-FMAU or 2′-fluoro-5-methyl-beta-L-arabinofuranoyl uridine is disclosed in U.S. Pat. Nos. 5,565,438, 5,567,688 and 5,587,362.
Adefovir or (9-[2-(phosphono-methoxy)ethyl]adenine also referred to as PMEA is disclosed in U.S. Pat. Nos. 5,641,763 and 5,142,051. The corresponding prodrug, referred to as adefovir dipivoxyl, is clinically approved as an orally acting anti-HBV agent.
U.S. Pat. Nos. 5,444,063 and 5,684,010 disclose the use of enantiomers of beta-D-1,3-dioxolane nucleoside to treat HBV.
U.S. Pat. No. 6,881,831 to lyer et al. discloses compounds comprising two or more deoxyribonucleotide and/or ribonucleotide monomers connected by internucleotide linkages for use in the treatment of HBV.
L nucleosides of different structures have been claimed as anti-HBV agents in filed applications WO 08/40164, WO/95/07287 and WO 00/09531.
Other anti-HBV agents claimed include: (1) beta-D-3′azido-2,3-dideoxy 5-fluorocytidine (Mahmoudian, Pharm Research 8, 1198-203, 1991; (2) 2′-beta-D-F-2′,3′-dideoxynucleoside analogs, Tsai et al., Biochem Pharmacol. 48, 1477-1481, 1994; (3) 5-carboximido-, or 5-fluro-2,3 unsaturated or 3′-modified pyrimidine nucleosides.
In addition to adefovir, a few nucleotide analogs have also been claimed to be anti-HBV agents. These include 9[1-phosphonomethoxycyclopropyl)methylguanine], PMCG and its dipivaloxyl prodrug, PMCDG and the trifluoromethyl analog, MCC-478. For a review, see: Iyer et al., Current Opinion in Pharmacol 5, 520-528, 2005.
Cyclic nucleoside phosphonate analogs and prodrug derivatives are also nucleotide analogs with anti-HBV activity. The corresponding phosphoramidate prodrug analogs are converted to the phosphonate derivative by presumably by esterase enzymes. For a review, see: Iyer et al., Current Opinion in Pharmacol., 5, 520-528, 2005.
The concept of using chemically modified drugs as prodrug analogs is an established paradigm in the pharmaceutical development of a number of different drugs. The prodrug strategies permit transient modification of the physicochemical properties of the drug in order to: (a) improve chemical stability, (b) alter aqueous solubility, (c) improve bioavailability (d) target specific tissues (e) facilitate synergistic drug combinations, (f) overcome first-pass metabolic effects, (g) serve as lipophilic carrier for hydrophilic drugs, and (h) serve as a chemical depot for sustained drug delivery.
A few prodrug strategies have been employed to improve bioavailability, to enhance liver tissue distribution and to improve antiviral potency. For example, modification of the phosphate group as the corresponding amino acid phosphoramidate results in more potent antivirals (Gudmundsson et al., Nucleosides, Nucleotides, 23, 1929-1937, 2004. Cahard et al., Mini Reviews Med Chem., 4,371-381, 2004. Glyceryl phosphate and phospholipid prodrugs of nucleosides have also been developed (Hostetler et al., Antimicrob Agents and Chemotherapy, 44, 1064-1069, 2000) to improve oral bioavailability. S-acylthioethyl (SATE) and cyclic salicyl derivatives (cyclosal) are other examples of prodrug derivativation of nucleosides and nucleotides (Peyrottes, et al., Mini Rev. Med. Chem., 4, 395-408, 2004) and Meier et al., Mini Rev Med Chem 4, 383-394, 2004. Other prodrug strategies include 4-arylsusbstituted cyclic 1,3-propanyl esters (HepDirect analogs) designed to undergo oxidative cleavage by liver enzymes to release the active nucleotide intracellularly (Erion et al., J. Am. Chem. Soc., 126, 5154-5163, 2004).
In general, all nucleosides need to be phosphorylated to nucleoside mono-, di-, and triphosphates before they can become inhibitors of HBV polymerase. Thus, nucleosides can be considered as prodrugs, which need to be activated in vivo. Since most nucleosides target viral polymerase and act by similar mechanism of action, there is potential for rapid emergence of resistance and occurrence of adverse events such as mitochondrial toxicity due to inhibition of human gamma polymerase. Another problem with antiviral therapy is viral rebound following cessation of therapy.
Prodrug strategies are also being applied in the case of oligonucleotides (18-30 mers), which are being developed as potentially novel class of therapeutic agents using technologies such as aptamers, antisense, ribozymes, RNA interference, and immunostimulation [For reviews see: (a) Szymkowski, D. E. Drug Disc. Today 1996, 1, 415; (b) Uhlmann E.; Peyman A. Chem. Rev. 1990, 90, 543 (c) Uhlenbech O. C. Nature 1987, 328, 596; (d) Zamore P. D. Science, 2002, 296, 1265; (e) Manoharan, M. Curr. Op. Chem. Biol. 2004, 8, 570; (f) Iyer, R. P.; Kuchimanchi, S.; Pandey, R. K. Drugs of the Future 2003, 28, 51 (g) Uhlmann, E.; Vollmer, J. Curr. Opin. Drug Discov. Devel. 2003, 6, 204].
Being highly charged, large molecular weight compounds, oligonucleotides have unfavorable physicochemical attributes for cell permeation by passive diffusion. Consequently, the design of prodrug analogs of oligonucleotides has mainly focused on the partial masking of some of their negatively charged backbone by bioreversible, lipophilic groups. Several such analogs have been synthesized and bioreversibility has been demonstrated in vitro. However, it appears that although the initial unmasking of one or two nucleotides take place rapidly, complete unmasking takes several hours or even days. For example, Iyer et al., prepared S-acyloxyalkyl derivatives of a mixed PO-PS oligonucleotide and found that in vitro, they could convert back to the parent oligonucleotide albeit slowly. A similar SATE prodrug strategy has been employed for oligonucleotide prodrugs. But, there has not been a demonstration of their in vivo potential either in terms of improved pharmacokinetics of oligonucleotides or enhanced biological activity. Also, there are no reports of in vivo oral bioavailability studies of oligonucleotide prodrugs or demonstration of in vivo biological activity.
Shorter chain oligonucleotides (less than 8-mers) with lesser number of charges and smaller molecular weight compared to 20-mer oligonucleotides represent a promising class of novel molecules with potential therapeutic and diagnostic properties. Indeed, recent reports suggest that mono-, di-, tri-, and short chain oligonucleotides possess significant biological activity that can be exploited for therapeutic applications.
However, the lack of oral, transdermal, and other non-invasive, patient-compliant delivery systems, coupled with inefficient cellular permeability, represents a significant hurdle in the therapeutic advancement of these molecules.